Many pharmacological effects of ethanol have been postulated to be mediated via GABA-containing neuronal systems in the CNS. The GABA receptor exists as part of a complex containing receptors for benzodiazepines (BDZ), barbiturates, and the chloride ion channel. To examine the effects of ethanol on this complex, we have studied the binding properties of 35-S-t-butylbicyclophosphorothionate (35-S-TBPS), a ligand that interacts with the regulatory site of the chloride ion channel in membranes of cortex and cerebellum of C57B1 mice in the presence and absence of ethanol. In vitro addition of ethanol produced a dose-dependent inhibition of TBPS binding in both cerebellum and cortex (IC-50 approximately 300 mM). At a concentration of ethanol that is physiologically attainable, i.e., 100 mM, there was substantial inhibition of TBPS binding. GABA and pentobarbital also inhibited the binding of 35-S-TBPS in cortex and cerebellum, and ethanol did not potentiate the actions of these agents. Our initial results indicate that after chronic in vivo ethanol administration, which produces functional tolerance and physical dependence, tolerance to the in vitro effects of ethanol on TBPS binding does not occur, and the effects of GABA and barbiturates are also unchanged. In contrast to the results with TBPS, chronic ethanol treatment of mice resulted in a reduced ability of pentobarbital, added to assays in vitro, to stimulate BDZ (3-H-Flunitrazepam) (Flu) binding in cortex and cerebellum. Similar results were found in animals that were chronically fed barbiturates. The results support the hypothesis that certain pharmacological effects of ethanol may result from interactions with the GABA-BDZ-barbiturate receptor complex. Chronic treatment with ethanol results in possible adaptive changes that occur at a site other than that originally influenced by ethanol, but that alter the function of the complex as a whole. These biochemical alterations may be associated with various aspects of tolerance, cross-tolerance, and physical dependence on ethanol.